Supplementary material
Figure Legends
Figure S1. The expression of non-spliced and mature FLC mRNA are comparable when the repression of
FLC is stable.
qRT-PCR was used to estimate the level of FLC non-spliced transcripts and FLC mRNA after
vernalization. Germinating seed were placed at 4°C for different times (1 day, 7 days etc) before
being transferred to 22°C. Plants were harvested when they had developed 6 leaves. The data
presented are an average of at least 4 biological replicates ± SE.
Figure S2. The nucleosomes in positions +1 and -1 show increased occupancy after a 4 week
vernalization treatment.
(a) Nucleosome-occupancy was measured in ColFRISf2plants grown for 15 days (NV) and then shifted
to 4°C for 2 (2WV) or 4 weeks (4WV). Recovery of DNA immunoprecitipated with an antibody
against histone H3 was measured by qPCR. Recovery of fragments from FLC was normalised against
AG-UTR. The data presented are an average of 6 biological replicates ± SE. *p < 0.05; **p < 0.01;
*** p < 0.001
(b) There was no change in occupancy of the nucleosomes surrounding the transcription start of
UBC after a 4 week cold treatment. Recovery of fragments from UBC was normalised against AGUTR. The data presented are an average of 3 biological replicates ± SE.
Figure S3. There is no change in histone acetylation in a vin3-4 mutant following vernalization.
Enrichment of H3K9Ac in FLC chromatin was measured by ChIP-qPCR assays in ColFRISf2 plants (a) or
vin3-4 (b) grown as at 22°C for 15 days (NV) or after 15 days at 22°C followed by 4 weeks at 4°C
(4WV). FLC amplicons were normalized against SAM and then against histone H3. The data
presented are an average of 3 biological replicates ± SE. *p < 0.05; **p < 0.01
Figure S4. There is no change in H3K27me3 in a vin3-4 mutant following vernalization.
Enrichment of H3K27me3 in FLC chromatin was measured in by ChIP-qPCR assays in ColFRISf2 plants
(a) or vin3-4 (b) grown as at 22°C for 15 days (NV) or after 15 days at 22°C followed by 4 weeks at
4°C (4WV). FLC amplicons were normalized against AG-UTR and then against histone H3. The data
presented are an average of 3 biological replicates ± SE. *p < 0.05
Figure S5. The composition of nucleosomes in FLC chromatin changes in response to vernalization
(a & c) HTR3::FLAG and (b & d) HTR9::FLAG was enriched in FLC chromatin in cold-treated plants.
Enrichment was measured by ChIP-qPCR assays in transgenic plants expressing HTR3::FLAG or
HTR9::FLAG grown as for Fig 2(b) for panels (a) & (b); for panels (c) & (d) plants were harvested after
4 weeks (4WV), or following an additional 14 days’ growth at 22°C (4WV + 14). FLC amplicons were
normalized against AG-UTR then against histone H3. Amplicon R2 lies within the FLC promoter, R9
spans the nucleation region, and R11 & R14 are located in intron1. The data presented are an
average of 6 biological replicates ± SE. *p < 0.05; ***p < 0.01